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Article

Endophytic Effect of the South African Beauveria bassiana Strain PPRI 7598 on the Population Growth and Development of the Russian Wheat Aphid, Diuraphis noxia

by
Lisemelo Francina Motholo
1,2,3,*,
Marde Booyse
4,
Justin Louis Hatting
1,5,
Toi John Tsilo
1,6,
Makhotso Lekhooa
3 and
Oriel Thekisoe
2
1
Agricultural Research Council-Small Grain Institute, Private Bag X29, Bethlehem 9700, South Africa
2
Unit for Environmental Sciences and Management, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
3
Preclinical Drug Development Platform, Faculty of Health Sciences, North-West University, Potchefstroom 2520, South Africa
4
Agricultural Research Council—Biometry, Private Bag X5013, Stellenbosch 7599, South Africa
5
Andermatt-PHP (Pty) Ltd., 19 Petrus Stroom Road, Dargle 3265, South Africa
6
Department of Life and Consumer Sciences, University of South Africa, Florida 1710, South Africa
*
Author to whom correspondence should be addressed.
Agriculture 2023, 13(5), 1060; https://doi.org/10.3390/agriculture13051060
Submission received: 13 April 2023 / Revised: 6 May 2023 / Accepted: 10 May 2023 / Published: 15 May 2023
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Abstract

:
The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is one of the main pests of small-grain cereal crops, including bread wheat, Triticum aestivum (Poaceae). In a series of glasshouse experiments, we evaluated the systemic effect of endophytic Beauveria bassiana strain PPRI 7598 on D. noxia biotype RWASA1 using three South African wheat cultivars, Gariep, Molopo, and Kariega. The objectives of the study were: (a) to determine the effect of endophytism on RWA reproduction and population growth, and (b) to assess the effect of the endophyte on aphid biomass and T. aestivum response to D. noxia herbivory using a damage rating index. Plant endophytic colonisation was confirmed before each trial using a B. bassiana-selective medium. Three independent trials were performed 10 days apart under glasshouse conditions. The effect of the endophyte-treated versus non-treated plants varied significantly in terms of net reproductive rate (R0) and the intrinsic rate of increase (rm) of the D. noxia population. Overall, the endophyte significantly reduced D. noxia R0 by approximately 14 nymphs/female and decreased the aphid mass by 13% in treated plants, whereas the mean aphid mass increased by 17% in control plants in all pooled cultivars. These findings demonstrated the endophytic potential of B. bassiana strain PPRI 7598 for suppression of D. noxia populations in RWASA1-susceptible cultivars. The integration of B. bassiana endophytism with host plant resistance may counteract biotype development and support a more sustainable approach towards RWA control in integrated pest management programmes.

1. Introduction

Wheat (Triticum aestivum L.) (Poaceae) is the second-most important cereal cash crop grown in South Africa (SA) after maize [1]. However, the country’s wheat production has lately suffered a 16% decline in grain production, from an average of 1.83 million tons in 2007/8 to 1.54 million tons in 2016/17 (South African Grain Laboratory [SAGL] report of 2018). Factors adversely affecting South African wheat production include, among others, diseases and insect pests, causing damage that seriously reduces farmers’ profits. As a result, growers have either shifted their focus to more profitable commodities or quit farming altogether [1]. Among the common insect pests of wheat, aphids (Hemiptera: Aphididae) are the most notorious and capable of causing considerable damage to this and other cereal crops, contributing greatly to yield loss [2].
Owing to their parthenogenetic reproduction and short generation time, aphid populations grow exponentially, and readily exceed the integrated pest management (IPM) economic threshold [3]. In South Africa, wheat cultivated under winter rainfall, summer rainfall, and irrigated conditions is prone to infestation by any of six cereal aphid species [4]. Of these, the Russian wheat aphid (RWA), Diuraphis noxia Kurdjumov (Hemiptera: Aphididae), is the most injurious, prompting research on host plant resistance and subsequent release of the first RWA-resistant cultivar, Tugela-DN, in 1992 [5]. However, with wheat monoculture practice, host plant resistance brings about selection pressure, and lead to development of resistance-breaking aphid biotypes [6,7].
To date, at least five RWA biotypes have been reported in the summer rainfall area of South Africa [8]. Clearly, measures to protect and sustain genetic control of RWA include the possibility of using endophytic fungi [9] to suppress aphid population growth and development. Locally (South Africa), wheat endophytism by the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) was demonstrated in a study by Motholo et al. [10]; also confirming the pathogenicity of that strain (PPRI 7598) to RWA following plant-host passage [11]. Although the in planta effect of endophytic B. bassiana has not been investigated against RWA on wheat, impaired aphid fitness was observed when challenged by (other) endophyte-infected grasses [12,13,14]. Cases of reduced insect fitness in a Beauveria–Poaceae association, involve the Acrididae [15,16], Noctuidae [17,18], and Scutelleridae [19].
By producing toxic substances (alkaloids) and other metabolites in the plant tissue, fungal endophytes can inhibit insect feeding and reduced growth rate at their feeding site [17,20,21]. For example, endophyte colonisation of cotton leaves by B. bassiana adversely affected the insects’ reproduction [15], infestation rate, and growth on grapevine plants relative to that of endophyte-free plants [22].
The mutualist benefit is understood to be attributed to the ability of the endophyte to alter the host plant’s nutritional quality [23] to secrete secondary metabolites (gibberellins and indole-acetic acid) [24] and to emit volatile compounds such as terpenes, flavonoids, alkaloids, quinines, cyclohexanes, and hydrocarbons. Some of the latter have been reported to be released in response to insect herbivory, thereby deterring insect feeding on endophytically colonised plants [25]. Several studies have confirmed the successful reduction of insect herbivory from piercing-sucking, boring, chewing, and leaf-mining pests residing on endophyte-colonised plants [9].
In our previous study, three South African dryland wheat cultivars (Tugela, Elands, and Gariep) and two irrigation-type cultivars (Kariega and Baviaans) were inoculated with B. bassiana PPRI 7598, which induced 71% plant growth improvement of wheat with endophytic establishment across all cultivars. These findings support the notion of employing a systemic biological approach as additional IPM component against RWA. Previously, topical applications of B. bassiana, in combination with host plant resistance, yielded around 65% control [26], although such applications are challenged by conidia being exposed to adverse environmental conditions (especially UV light), insufficient contact with the target and ‘loss’ of conidia from the insect cuticle during ecdysis. Moreover, the five other cereal aphid species (greenbug Schizaphis graminum R., English grain aphid Sitobion avenae F., bird cherry-oat aphid Rhopalosiphum padi L., corn aphid R. maidis F., and rose grain aphid Metopolophium dirhodum W.) are not believed to be affected by the resistance genes employed against RWA; an endophytic approach, however, may also impair the fitness of those species.
In the current study, we evaluated the systemic effect of endophytic B. bassiana strain PPRI 7598 against a D. noxia biotype (RWASA1) on three SA T. aestivum cultivars, Gariep, Molopo, and Kariega (Gariep being resistant to RWA). Specifically, the purpose of this study was: (a) to determine the effect of the endophyte on RWA reproduction and population growth, and (b) to assess the effect of the endophyte on aphid biomass and T. aestivum response to D. noxia herbivory using a damage rating index [27].

2. Materials and Methods

Three wheat cultivars, inoculated with the endophytic B. bassiana strain PPRI 7598, and untreated controls were infested with D. noxia (RWASA1) under glasshouse conditions. The effects of the two treatments were compared across the three trails.

2.1. Experimental Design

In experiment 1, a series of three independent trials were conducted under glasshouse conditions. For each trial, the experimental design was a randomised complete block with 10 replicates. The treatment design was a split-plot: the main plot was three cultivars (Molopo, Gariep, and Kariega) and subplots contained plants inoculated with the endophyte B. bassiana. Sixty plants (10 plants × 2 treatments × 3 cultivars) per trial were assayed against D. noxia infestation.
In experiment 2, three sets of trials were conducted. For each trial, the experimental design was a randomised complete block with three replicates. The treatment design consisted of two factors, that is, three cultivars (Molopo, Gariep, and Kariega) and B. bassiana with two treatments (treated and untreated). The experiment consisted of 90 plants (3 cultivars × 5 replicates × 3 trials × 2 treatments).

2.2. Aphid Rearing

The RWASA1 colonies were reared in the ARC-Small Grain’s insectary unit, in Bethlehem, SA. An aphid clone was established from one adult female, which was maintained on a single intact wheat plant (cv.Tugela) for 24 h in the laboratory at room temperature. The adult aphid was then removed, leaving a clone of first filial generation (F1) nymphs on the plant. Each of the F1 nymphs in the first instar was transferred to a single wheat seedling and reared in a gauze-covered cage in a glasshouse at 22 ± 3 °C, 70 ± 4% relative humidity (RH) in natural light [28]. Aphids were monitored until they reached the adult reproductive stage and produced the F2 generation for use in the trial. These aphids were further monitored for one week before they were transferred to experimental and control plants (using a fine camel hairbrush) at the four-leaf growth stage.

2.3. Endophytic Fungal Strain

The B. bassiana fungal strain PPRI 7598, found to be virulent against the RWASA1 in previous studies [11] and showing potential to colonise wheat seedlings following seed treatment [28], was used in this study. The fungal strain was cultured on B. bassiana-selective medium amended with 0.55 g L−1 of dodine (guanidine) and 0.005 g L−1 of chlortetracycline (Sigma–Aldrich, Burghausen, Germany) [29]. Fungal cultures were incubated in the dark at 25 ± 1 °C and 60 ± 10% RH. Conidia were harvested from 14-day-old cultures (using a sterile scalpel and camel hairbrush) and suspended in distilled water with 0.01% Break Thru® surfactant (polyether–polymethylsiloxane copolymer surfactant, Goldschmidt Chemical Corporation, Virginia, VA, USA). The fungal suspension was then vortexed for at least 5 min to produce a homogeneous stock suspension, which was adjusted to 108 conidia mL−1 concentration using a Nikon Optiphot light microscope (Nikon, Minato, Japan) and Improved Neubauer haemocytometer (Neubauer, Germany).

2.4. Wheat Seed Inoculation and Planting

Seeds of three T. aestivum cultivars (Molopo, Gariep, and Kariega) were obtained from the ARC-Small Grain’s Germplasm collection. Seeds were surface sterilised based on a standard procedure with minor modifications [30], after which they were dried in a laminar flow cabinet for 30 min and later immersed in a B. bassiana suspension with concentration of 108 conidia mL−1. Control seeds were soaked in 5 mL of sterile distilled water with 0.01% Break Thru® surfactant. Twenty seeds were soaked for 18–24 h in 5 mL spore suspension with occasional aeration while maintained on an orbital shaker at room temperature [31]. Inoculated and control seeds were later dried on a sterile paper towel in a laminar flow atmosphere for three hours prior to planting. Seeds were first plated between moist filter papers (Lasec, Cape Town, South Africa) in 90 mm Petri dishes, and incubated in the dark at 25 ± 1 °C to enhance germination. Seedlings were later transplanted singly in cones (100 mL), which contained a sterile soil/sand substrate (3:1 volume ratio) and sterilised in an autoclave at 121 °C for 15 min. Plants were maintained in water-filled trays under greenhouse conditions (22 ± 3 °C, 70 ± 4% RH) under natural light for 21 days. Sixty experimental plants (3 cultivars × 10 replicates × 2 treatments) each were later infested with one of the age-synchronised RWA adults (8 days old).

2.5. In Vivo Bioassays

2.5.1. Experiment 1: Effect of the Endophyte on Aphid Reproduction and Population Growth

The RWA colony/clone was established in the greenhouse under similar conditions as above (see § 2.4). The experimental design was as described in § 2.1. The same greenhouse conditions applied to experimental (endophyte-treated) and control (endophyte-free) plants. The experiments were performed on un-infested intact plants. Separate trials consisted of 60 individual plants at the four-leaf growth stage, each infested with one F2 generation aphid at the reproductive adult stage (8 days old). All plants with aphids were enclosed in clear ventilated plastic bottles (16 cm × 9 cm), inverted to cover plants and to prevent aphids from escaping or mixing between treatments. Aphid reproduction based on the number of nymphs born was recorded daily from each plant for 15 days. Following enumeration, all newly born nymphs were removed from plant leaves while adults remained. Net reproductive rate (R0) and mean generation time (T) were estimated across the three cultivars and treatments, to determine the intrinsic rate of aphid population increase (rm), which was calculated using the formula [32]:
∑ e − rx   ( l x × m x ) = 1 ,
where x is the time increment (24 h), l x is the probability of being alive on day   x , m x is the average birth rate on day x , and r is the intrinsic rate of increase. Experiments were repeated three times, 7 days apart.

2.5.2. Experiment 2: Effect of the Endophyte on Aphid Biomass and T. aestivum Response towards RWA Herbivory

The systemic effect of plant endophytic colonisation was evaluated on the three wheat cultivars maintained under greenhouse conditions of 22 °C (day) and 15 °C (night) with natural light. There were 30 plants per cultivar with two treatments per batch (endophyte treated and untreated). All 90 plants (3 cultivars × 5 replicates × 3 blocks × 2 treatments) were organised in a randomised design on a 94-cone seed tray placed in water-filled trays. A tray with all plants at the 4-leaf growth stage was infested with 450 apterous D. noxia, weighing ~0.05016 g (mixed instars taken from the greenhouse colony), giving an average infestation of five aphids per plant [33]. The aphid population per plant was harvested and weighed to determine the RWA biomass across the cultivars and treatments 21 days after infestation. Plants were also evaluated for their response to RWA herbivory and rated for D. noxia damage. The severity of infestation was assessed according to a damage rating system using a 1–10 scale per plant [27]. This index was used to indicate the feeding intensity and damage levels caused by RWA herbivory on both treated and control plants. Population growth was determined on day 22 of aphid infestation.

2.5.3. Confirmation of Plant Endophytic Colonisation

Endophytic colonisation of wheat plants was determined at 6 weeks post-inoculation, that is, at the end of experiment 1. Two detection methods based on culturing (fungal re-isolation) and species-specific PCR diagnostic techniques were deployed to confirm B. bassiana colonisation of wheat seedlings across the three selected cultivars. At the end of each trial, all 60 experimental plants (treated and control) were harvested. Adapted from Castillo-Lopez’s study [34], two leaves from each plant were surface sterilised [10,35] and excised into two longitudinal halves using a sterile scalpel. One half was cut to 1 cm lengths and plated on B. bassiana-selective medium (as in § 2.5.3). Unlike the previous study [10], evaluation for endophytic colonisation of seedlings focused on leaves only (the RWA feeding site). Cultures were evaluated for fungal growth from day 7 to day 10 of incubation. The colonisation rate was calculated as the number of colonised segments divided by the total number of segments examined, multiplied by 100 [36]. However, data were captured only to confirm positive endophytic colonisation of plants and not for statistical analysis.
The other half of each leaf was freeze-dried (Christ® Alpha 2–4 LD plus, Mechatechsystems, Bristol, UK) and DNA extracted using the CTAB protocol [37]. Diagnostic PCR analysis was performed using B. bassiana-specific primers [34] to amplify the endophyte DNA. The PCR amplicon was visualised on 1.5% agarose gel using gel electrophoresis under a UV illuminator (Molecular Imager® Gel Doc™ XR System, Bio-Rad, SA, Hercules, CA, USA). The fragment size of the amplified DNA from endophyte-treated plants was confirmed based on the positive control strain (PPRI 7598) size.

2.6. Statistical Analysis

The homogeneity of trial variances was verified using Levene’s test [38]. The normality of the standardized residuals was confirmed using the Shapiro–Wilk test [39]. The data of the combined trials were subjected to analysis of variance (ANOVA) using General Linear Models Procedure (PROC GLM) of SAS software (Version 9.4; SAS Institute Inc., Cary, NC, USA). Where repeated measurements on the same experimental unit were taken, a split-plot analysis of variance with days as sub-plot factor was performed according to the method described by Montgomery (2005) to compare treatment differences over time [40]. Fisher’s protected least significant difference (LSD) was calculated at the 5% level to compare treatment means [41].

3. Results

3.1. Effect of the Endophyte on RWA Reproduction and Intrinsic Rate of Population Increase

Significant effects were observed between the two plant treatments, that is, endophyte-treated versus endophyte-free (+endophyte and -endophyte, respectively) at p ≤ 0.05 (Table 1). The effect of the two treatments varied significantly in terms of net reproductive rate (R0) and intrinsic rate of increase (rm) of the RWA population over the average of 6.5 days’ mean generation time (T) across the three trials (Table 1).
A significant difference in RWA population growth (R0 and rm) was observed between trials (p < 0.05; LSD = 0.6401 and p < 0.05; LSD = 0.0488, respectively). Trial 3 recorded the lowest intrinsic rate of 0.372 ± 0.022 nymphs/female/day on endophyte-treated plants compared to the control, 0.462 ± 0.010 (Table 2). An endophyte treatment in plants negatively affected RWA R0 and reduced the population growth by 14 nymphs/female/day compared to the endophyte-free plants (control) (Table 2). However, the results indicated a poor correlation between the R0 and rm across trials, although treatments were significant at p < 0.0001. Overall, the endophyte significantly reduced the RWA population on treated plants over the control (Table 2).
The results suggest that there were no interactions between trials and cultivars. However, an interaction among cultivars and treatment was noted. The cultivars’ response to the treatment influenced the RWA population increase significantly (p < 0.05). An effect of the endophyte on RWA settlement on different cultivars was noted at the 5% test level, significantly reducing D. noxia R0 by approximately 14 nymphs/female (p < 0.05) for all cultivars (Table 3). While Kariega and Molopo are known to be susceptible to D. noxia RWASA1, the presence of the endophyte induced some level of resistance in Molopo towards D. noxia. Such resistance adversely affected aphid R0 levels on Molopo and Gariep (the resistant cultivar). This indicated that Kariega remained susceptible to D. noxia even in the presence of the endophyte; hence, it supported aphid reproduction to an average of 27.39 ± 0.36 (Table 3).

3.2. Effect of the Endophyte on Aphid Mass and T. aestivum Response to RWA Herbivory

On the assumption that the total initial mass of aphids (0.0502 g) halved to equally infest both groups of endophyte-treated and control plants (0.0251 g each), the aphid masses differed significantly (p < 0.05) between the treatments based on suitability of plants for foraging. Significantly higher aphid masses (p < 0.05) were observed on endophyte-free plants—higher on the RWASA1-susceptible cultivars (Molopo and Kariega) than on resistant Gariep. Thus, the endophyte had a significant effect on D. noxia mean mass, reducing it in the case of Molopo by 43% and 29% in Gariep when compared with the endophyte-free plants (Figure 1). However, the Kariega cultivar became equally suitable for foraging by this pest in both treatments.
Overall, the mean aphid mass increased by 17% from the initial 0.02501 g in control plants to the final mass of 0.0294 g; whereas the endophytes significantly decreased the aphid mass by 13% over the control, across all cultivars pooled (p = 0.05; LSD = 0.0065) (Figure 2). However, the damage rating varied significantly (p < 0.05) across the three cultivars, the highest being in the case of Molopo (9) followed by Kariega and Gariep (8 and 5, respectively) (Figure 3). Gariep was the only RWASA1-resistant cultivar, although there was no significant difference among the treatment levels across the three cultivars at p ≤ 0.0001.

3.3. Confirmation of Plant Endophytic Colonisation

Physical recovery of the endophytic strain from the inoculated plants (Figure 4A) was performed using a B. bassiana-selective medium. All plant parts from the endophyte-inoculated plants showed a B. bassiana outgrowth when cultured in a selective medium, indicating that they had been colonised endophytically by the B. bassiana strain PPRI 7598 (Figure 4B). No B. bassiana outgrowth was observed either on the control samples or on plated surface sterilisation final rinse water.

4. Discussion

In this study, the effect of the endophyte B. bassiana strain PPRI 7598 on D. noxia was tested for the first time on three wheat (T. aestivum) cultivars (Gariep, Molopo, and Kariega) in South Africa. Aphid population growth, in the presence and absence of an endophyte, was evaluated in terms of the two life-table parameters [net reproductive rate (R0) and the intrinsic rate of increase (rm)] in planta. This study showed increasing D. noxia population rates (R0 values) in control plants when compared to B. bassiana-treated plants. In the previous studies, Gurulingappa et al. reported similar results whereby B. bassiana reduced the fecundity rate of the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), on treated plants [14], whereas in Castello-Lopez et al.’s study the endophyte negatively affected A. gossypii reproduction on B. bassiana-treated plants under both greenhouse and field conditions [34], both in planta. As highlighted in the previous studies [30,42], poor aphid performance could be attributed to the production of toxic alkaloids induced by the endophyte’s presence in host plants. Although rm did not differ significantly (p ≤ 0.0001) across all cultivars tested, the highest intrinsic rate (suggesting a rapid population growth rate) recorded in trial 1could have been due to the highest settlement of D. noxia on the Kariega cultivar relative to the two other cultivars. However, these results demonstrated a poor correlation between R0 and rm, although treatments were significant at p < 0.05 (Table 2).
Following a previous study conducted in South Africa by Jankielsohn [28], among three aphid biotypes (RWASA1, RWASA2, and RWASA3) tested, RWASA1 showed a positive intrinsic rate of increase on different cereal host plants. Although the endophyte was not involved in this investigation, RWASA1′s intrinsic rate of population increase was higher in the susceptible cultivar (Tugela) compared to the resistant Tugela Dn. In line with these results, RWASA1 demonstrated a similar trend of the highest intrinsic rate for Kariega over Gariep, even in the presence of the endophyte (p = 0.05; LSD = 3.168), indicating varying attraction to this aphid.
In our study, the highest D. noxia population was observed on Kariega, suggesting that this cultivar might be the most suitable host for this insect pest. Consequently, this resulted in an increased D. noxia R0 value (27.39 ± 0.36), coupled with increasing aphid masses (although not significant at p < 0.05) on endophyte-treated and endophyte-free plants (0.0374 g and 0.0359 g, respectively), as compared to the two other cultivars. This indicates that the endophyte-secreted metabolites did not alter palatability and aphid feeding preference in this cultivar. High plant palatability to sucking insects has been investigated in various studies [43,44,45] and has been found to result in high herbivore population densities [46], which gives a reason for the relatively high infestation population observed on Kariega. Greater plant palatability supports increased herbivore growth and feeding pressure due to weaker plant defence against insect herbivores, particularly at high relative to low altitudes [46], meaning in the cold winter regions of South Africa. Although trials were conducted under glasshouse conditions, Kariega could have attained genetic adaptation to thrive better in cold winter regions, hence being the most preferred susceptible host by D. noxia.
Several studies performed on aphids reared on different wheat cultivars indicated that impaired performance and reproduction are related to variable concentrations of allelochemicals such as hydroxamic acids, phenolic compounds, and indole alkaloids [47,48,49]. Hydroxamic acids are secondary metabolites of wheat that boost the cereal’s resistance to aphids [50] and are known to prolong aphid development and reduce fecundity as well as the intrinsic rate of their natural increase [51]. In this case, varying aphid masses found in Gariep’s endophyte-treated (0.0136 g) and endophyte-free plants (0.0192 g) suggest that both the hydroxamic acids and secondary metabolites induced by the endophyte had a deterring effect on D. noxia feeding. The production of alkaloids by the endophytes increases the host plant’s competitive ability and resistance to biotic stressors [52], particularly insect herbivores. In support of this point, the evidence of endophyte-mediated negative effects on herbivores’ foraging was observed on the resistant cultivar, Gariep, in our study. This confirms that integration of both the endophyte and plants’ defence mechanisms significantly improved plant resistance, lowering the aphid masses on endophyte-treated plants by 26% (p < 0.05). However, the parity of the aphid masses between the cultivars Molopo (susceptible) and Gariep, in the presence of the endophyte, was observed. This may indicate that the endophyte had potentially improved host resistance to aphid herbivory in Molopo.
Resistance of the host plant to insect herbivory protects the plant against adverse conditions associated with pest damage. We recorded a distinctive damage rating by the aphid biotype RWASA1 between Gariep, a resistant cultivar, and the susceptible cultivars Molopo and Kariega in the presence of the endophyte. The levels of damage in these cultivars varied significantly, ranging from chlorotic leaf streaking to leaf rolling or manifesting both effects, depending on the degree of plant susceptibility and damage caused [26]. Similar results were reported in a previous study in which the damage levels caused by D. noxia were characterised on a US winter wheat cultivar [53]. Notably, leaf rolling reduces the host plant’s photosynthetic area while affording an optimum environment for aphid reproduction in the leaf whorl [54]. This condition was observed in our study, where Molopo and Kariega recorded the highest damage scores (9 and 8, respectively) with relatively high aphid populations enclosed inside rolled leaves.
The assumption made when measuring plant damage was that aphids would be distributed evenly, giving an average of five aphids per plant pre-trial [26]. However, the random distribution of aphids of mixed instars on plants could present various possibilities for plant infestation. This arrangement might have afforded aphids a wider choice to select plants according to their preference for susceptible plants, possibly avoiding settling on resistant plants. In this case, it could be assumed that the majority of adult aphids started producing progeny at the beginning of the experiment, rapidly increasing the aphid mass on susceptible plants. Alternatively, immature aphids on endophyte-treated plants might have stopped feeding, resulting in delayed reproduction as compared to those on control (endophyte-free) plants. Previous studies presented several mechanisms by which endophytic fungi reduce insect herbivore damage potential, but enhance a reduction in the insect development rate [30] as a result of feeding deterrence [55,56], retardation of insect growth, and reduced survival and oviposition [42,57]. Otherwise, aphid feeding (characterised by the secretion of saliva in the plant phloem) could have exacerbated plant damage by changing the host plant’s physiology [58].
In the majority of studies associated with endophyte-herbivore interactions, the physiological responses of resistant and susceptible cereals to D. noxia feeding have been reported, particularly of wheat [59,60,61]. Such research focused on chlorophyll and protein contents, chlorophyll fluorescence, gas exchange, and molecular pathways [54]. In one previous investigation, susceptible plants demonstrated a change in chlorophyll content with observable leaf chlorosis relative to resistant plants [62]; similar observations were recorded in our study. These observations are supported by the fact that D. noxia can alter the amino acid profile of the target plants’ phloem; as a result, the susceptible wheat cultivar in this study might have encountered nutrient alteration as a result of D. noxia feeding [63]. Consequently, this activity induced a retarded growth pattern observed in susceptible wheat cultivars in our study. The damage associated with D. noxia feeding could have contributed to significantly higher damage levels recorded on Molopo (Figure 3), despite the low aphid mass response recorded on this cultivar (Figure 1). However, the effect of D. noxia feeding did not affect the nutrient profile, thereby resulting in lower damage levels in the resistant cultivar Gariep than in Molopo [63].
Mechanisms underpinning the endophyte-mediated nutritional quality of plants and production of secondary metabolites are known to affect insect feeding on endophyte-colonized plants [64]. However, it was reported with the bird cherry-oat aphid, R padi, that resistant wheat plants may trigger some antibiosis-based responses against the aphid, by deploying mechanisms that inhibit aphid induction of defensive compounds in wheat seedlings [7,65]. Apparently, integration of the host plant’s resistance and endophyte-induced systemic resistance could work as an alternative IPM approach to suppress the activity of RWASA1 and other biotypes from adapting antibiosis and/or antixenosis responses, which could break Gariep’s resistance. This effect of the changes induced by an endophyte in a host plant could explain the significant differences in damage ratings observed between susceptible and resistant wheat cultivars in our study.
The use of surface sterilisation procedures to rid plant parts of associated epiphytes may have underestimated the colonisation rates owing to diffusion of chemicals into these parts [66,67], particularly leaves. Although the laceration of wheat leaves could have contributed to the low colonisation results in our study, we argue that the surface sterilisation method could have affected the sterilised material before a culture-based confirmation of endophytic colonisation was established [68]. Additionally, since the cutting of leaves (during the evaluation of endophyte colonisation) damages a plant, the subsequent production of allelochemicals around lacerated parts, including leaves, could suppress the existing plant endophytes [69], thereby resulting in poor detection of the target endophyte on a B. bassiana-selective medium.

5. Conclusions

This study indicates that the endophytic B. bassiana strain PPRI 7598 can improve wheat plants’ resistance to D. noxia in both resistant and susceptible South African cultivars. The integration of B. bassiana endophytism with host plant resistance may counteract biotype development, alleviate crop losses caused by damage from sucking insects, and regulate insect populations and infestations on crops by reducing the aphid’s reproduction and fecundity rates. More broadly, the ability of fungal endophytes to improve a plant’s systemic resistance to insect herbivory brings a new dimension worth further exploration in agriculture to boost crop yields, especially under conditions of climate change. The use of endophytes could be a suitable replacement for short-term pest control measures through topical applications when established in plants. This suggests formulating a sustainable and cost-effective IPM approach for the management of D. noxia, as well as other common cereal crop pests, on T. aestivum.

Author Contributions

Conceptualisation, J.L.H., O.T., T.J.T. and L.F.M.; Methodology, J.L.H. and O.T.; Formal analysis, M.B.; Funding acquisition, T.J.T. and O.T.; Supervision, J.L.H., T.J.T. and O.T.; Resources, T.J.T. and O.T.; Writing—original draft preparation, L.F.M., J.L.H. and O.T.; Writing—review and editing, J.L.H., T.J.T., O.T. and M.L. All authors have read and agreed to the published version of the manuscript.

Funding

The project was supported by the Professional Development Programme of the Department of Science and Technology (now known as the Department of Science and Innovation (DSI) with the National Research Foundation (DST-NRF) (Grant No.: 96715)), and the Agricultural Research Council of South Africa-Small Grain (ARC-SG).

Institutional Review Board Statement

This study was approved as a no risk study by scientific committee of the Integrated Pest Management of the Unit for Environmental Sciences and Management of North-West University. The study did not involve humans or higher animals, but only insects (lower invertebrate animals). Therefore, ethical review and approval were waived for this study.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to thank all the institutions that supported this study, namely: ARC-SG; NWU, and DST-NRF for funding this study project.

Conflicts of Interest

The authors declare no conflict of interest.

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Agriculture 13 01060 g001 550
Figure 1. Distribution of the mean aphid mass at the end of the experiment (p = 0.05; LSD = 0.0113). The indices -Endophyte denotes treatment without the endophyte (endophyte-free) and +Endophyte means treatment with the endophyte (endophyte-treated). The total initial aphid mass was calculated as 90 plants × 5 aphids × RWASA1 mass (0.000111475) = 0.0502 g. Bars (means ± SEM) marked with different letters indicate significant differences at the p = 0.05; LSD = 0.0065.
Figure 1. Distribution of the mean aphid mass at the end of the experiment (p = 0.05; LSD = 0.0113). The indices -Endophyte denotes treatment without the endophyte (endophyte-free) and +Endophyte means treatment with the endophyte (endophyte-treated). The total initial aphid mass was calculated as 90 plants × 5 aphids × RWASA1 mass (0.000111475) = 0.0502 g. Bars (means ± SEM) marked with different letters indicate significant differences at the p = 0.05; LSD = 0.0065.
Agriculture 13 01060 g001
Agriculture 13 01060 g002 550
Figure 2. The mean aphid mass for the endophyte-treated and control plants in all cultivars pooled. The indices −Endophyte denotes treatment without the endophyte (endophyte-free) and +Endophyte means treatment with the endophyte (endophyte-treated). Bars (means ± SEM) marked with different letters indicate significant differences at the p = 0.05; LSD = 0.0065.
Figure 2. The mean aphid mass for the endophyte-treated and control plants in all cultivars pooled. The indices −Endophyte denotes treatment without the endophyte (endophyte-free) and +Endophyte means treatment with the endophyte (endophyte-treated). Bars (means ± SEM) marked with different letters indicate significant differences at the p = 0.05; LSD = 0.0065.
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Agriculture 13 01060 g003 550
Figure 3. Mean damage rating analysis for the three cultivars induced by D. noxia, RWASA1 at p < 0.05: Description of the Russian wheat aphid damage symptoms used for scoring using a 1–10 damage rating system. Adapted from [27]. * –, no response; X, damage rating.
Figure 3. Mean damage rating analysis for the three cultivars induced by D. noxia, RWASA1 at p < 0.05: Description of the Russian wheat aphid damage symptoms used for scoring using a 1–10 damage rating system. Adapted from [27]. * –, no response; X, damage rating.
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Figure 4. (A) Endophyte-inoculated plants raised as one plant per container. Each plant was maintained under glasshouse conditions for 15 days after infestation with one adult aphid (D. noxia). (B) Evaluation of plant colonisation from different plant sections of leaves, stems, and roots. Isolation of endophytic fungus as observed on colonised plant sections plated on a B. bassiana selective medium [29]. Photograph: LF Motholo, ARC-Small Grain.
Figure 4. (A) Endophyte-inoculated plants raised as one plant per container. Each plant was maintained under glasshouse conditions for 15 days after infestation with one adult aphid (D. noxia). (B) Evaluation of plant colonisation from different plant sections of leaves, stems, and roots. Isolation of endophytic fungus as observed on colonised plant sections plated on a B. bassiana selective medium [29]. Photograph: LF Motholo, ARC-Small Grain.
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Table
Table 1. Representation of different life-table parameters that influenced D. noxia population growth in different trials and treatments under glasshouse conditions.
Table 1. Representation of different life-table parameters that influenced D. noxia population growth in different trials and treatments under glasshouse conditions.
TrialLife-Table Parameters
Net Reproductive Rate (R0)Mean Generation Time (T) (Days)Intrinsic Rate of Increase (rm)
129.65 ± 0.32a 1 (1) 26.0542 ± 0.47b (3)0.560 ± 0.13a (1)
224.52 ± 0.28b (2)6.2018 ± 0.59b (2)0.504 ± 0.15b (2)
321.37 ± 0.41b (3)7.3061 ± 0.53a (1)0.417 ± 0.16c (3)
Grand mean25.18 ± 2.416.5207 ± 0.390.494 ± 0.04
LSD0.053.5920.51712.0049
TreatmentR0Trm
−Endophyte 31.39 ± 0.24a (1)6.759 ± 0.42a (1)0.517 ± 0.12a (1)
+Endophyte 17.21 ± 0.33b (2)6.282 ± 0.69b (2)0.471 ± 0.20b (2)
Grand mean24.3 ± 7.096.521 ± 0.240.494 ± 0.02
LSD0.051.82930.38240.0282
1 Means ± SEM within columns followed by the same letter are not significantly different at the 5% test level. 2 Performance ranking within a given life-table parameter (column) in brackets. −Endophyte denotes treatment without the endophyte (endophyte-free). +Endophyte means treatment with the endophyte (endophyte-treated).
Table
Table 2. The effect of the treatment (endophyte-treated versus endophyte-free or control) on D. noxia population growth in different trials.
Table 2. The effect of the treatment (endophyte-treated versus endophyte-free or control) on D. noxia population growth in different trials.
TrialReproduction Rate (R0)Intrinsic Rate (rm)
+Endophyte−Endophyte+Endophyte−Endophyte
122.33 ± 0.269a 1 (1) 236.97 ± 0.230a (1)0.557 ± 0.023a (1)0.563 ± 0.012a (1)
217.67 ± 0.182b (2)31.37 ± 0.240b (2)0.483 ± 0.020bc (2)0.526 ± 0.018ab (2)
314.33 ± 0.229b (3)28.400 ± 0.201b (3)0.372 ± 0.022d (3)0.462 ± 0.010c (3)
* G. mean18.11 ± 2.3232.25 ± 2.510.471 ± 0.050.517 ± 0.03
LSD0.050.64010.0488
1 Means ± SEM within columns followed by the same letter are not significantly different at the 5% test level. 2 Performance ranking within a given life-table parameter (column) in brackets. −Endophyte denotes treatment without the endophyte (endophyte-free). +Endophyte means treatment with the endophyte (endophyte-treated). * G. mean denotes the grand mean.
Table
Table 3. The effect of the treatment (endophyte-treated and endophyte-free) on D. noxia population growth across cultivars.
Table 3. The effect of the treatment (endophyte-treated and endophyte-free) on D. noxia population growth across cultivars.
CultivarReproduction Rate (R0)
+Endophyte−EndophyteAverage
Kariega 20.64 ± 0.3a 1 (1) 234.15 ± 0.268a (1)27.39 ± 0.36a (1)
Molopo16.65 ± 0.234b (2)31.11 ± 0.263ab (2)23.88 ± 0.37b (2)
Gariep16.34 ± 0.167b (3)28.92 ± 0.191b (3)21.63 ± 0.33b (3)
* G. mean17.88 ± 1.3831.39 ± 1.5224.3 ± 1.68
LSD0.053.1683.1437
1 Means ± SEM within columns followed by the same letter are not significantly different at the 5% test level. 2 Performance ranking within a given life-table parameter (column) in brackets. −Endophyte denotes treatment without the endophyte (endophyte-free). +Endophyte means treatment with the endophyte (endophyte-treated). * G. mean denotes the grand mean.
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Motholo, L.F.; Booyse, M.; Hatting, J.L.; Tsilo, T.J.; Lekhooa, M.; Thekisoe, O. Endophytic Effect of the South African Beauveria bassiana Strain PPRI 7598 on the Population Growth and Development of the Russian Wheat Aphid, Diuraphis noxia. Agriculture 2023, 13, 1060. https://doi.org/10.3390/agriculture13051060

AMA Style

Motholo LF, Booyse M, Hatting JL, Tsilo TJ, Lekhooa M, Thekisoe O. Endophytic Effect of the South African Beauveria bassiana Strain PPRI 7598 on the Population Growth and Development of the Russian Wheat Aphid, Diuraphis noxia. Agriculture. 2023; 13(5):1060. https://doi.org/10.3390/agriculture13051060

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Motholo, Lisemelo Francina, Marde Booyse, Justin Louis Hatting, Toi John Tsilo, Makhotso Lekhooa, and Oriel Thekisoe. 2023. "Endophytic Effect of the South African Beauveria bassiana Strain PPRI 7598 on the Population Growth and Development of the Russian Wheat Aphid, Diuraphis noxia" Agriculture 13, no. 5: 1060. https://doi.org/10.3390/agriculture13051060

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